An important role for the activation peptide domain in controlling factor IX levels in the blood of haemophilia B mice

Abstract

The factors responsible for the removal of injected factor IX (fIX) from the blood of individuals with haemophilia B are only partly understood, and may include binding to endothelial or subendothelial sites, passive extravasation related to size or charge, or interactions requiring fIX activation. To investigate these issues, we have produced and characterised recombinant fIX proteins with amino acid changes: delta155-177, an internal deletion which removes most of the activation peptide while retaining the activation cleavage sites; S365A, which inactivates the serine protease activity of fIXa; and K5A, previously shown to eliminate fIX binding of endothelial/subendothelial collagen IV. All proteins were expressed in stably transfected HEK 293 cells, purified by immunoaffinity chromatography, and compared to the wild type HEK 293-derived protein (fIX (WT)). Mutant fIX proteins K5A and delta155-177 exhibited 72 and 202% of the specific activity of fIX (WT), respectively; S365A was without activity. Following intravenous injection in haemophilia B (fIX knockout) mice, recoveries did not differ for fIX (WT) and delta155-177, but were higher for K5A and S365A. The terminal catabolic half-life of delta155-177, alone among the mutants, was increased, by 45% versus fIX (WT). Nine hours post-injection, the observed areas under the clearance curve (AUCs) of delta155-177 and K5, but not S365A, were elevated 2-fold. delta155-177 was equally effective as fIX (WT) in reducing blood loss following tail vein transection in haemophilia B mice. Our results suggest that deletion of the multiple sites of fIX post-translational modification found within the activation peptide eliminated important fIX clearance motifs.

Targeted Inhibition of Serotonin Type 7 (5-HT7) Receptor Function Modulates Immune Responses and Reduces the Severity of Intestinal Inflammation

“…Tail vein bleeding model. Tail bleeding tests were done according to a published method (46). In brief, mice were anesthetized with isoflurane (Abbott), and the tail tip (0.7-1.0 cm) was incised using a fresh scalpel.…”

“…Tail bleeding times were recorded as the time that it took for mice to stop bleeding. Shed blood collected on filter papers was eluted in water and quantified using OD 405 and OD 490 and a standard curve as previously described (46).…”

Prolonged half-life and preserved enzymatic properties of factor IX selectively PEGylated on native N-glycans in the activation peptide

“…11 Various approaches have been pursued to extend the half-life of coagulation factors including genetic fusions to albumin and the Fc fragment of IgG as well as chemical modification by hydrophilic polymers such as polyethylene glycol (PEG). [12][13][14][15] The prolonged circulation time endowed by the latter approach is believed to arise from a reduction in the efficiency of various elimination processes such as renal excretion, receptor mediated uptake, and proteolytic degradation. 16 With several PEGylated protein-based therapeutics on the market since 1990, the efficacy and safety of this technology has undergone extensive clinical validation.…”

Fusion to Human Serum Albumin Extends the Circulatory Half-Life and Duration of Antithrombotic Action of the Kunitz Protease Inhibitor Domain of Protease Nexin 2

“…Acid-bound radioactivity was determined from serial plasma samples. Initial and terminal half-lives were determined using a two compartment model and curve-stripping as previously described [28, 29]. In some experiments radiolabelled protein preparations were combined with purified RAP or GST (0.15 mM final concentration) prior to injection, as previously described [30].…”